PROJECT SUMMARY The goal of my research program is to understand how cells detect and degrade aberrant RNAs, and how this surveillance process shapes gene expression. Nonsense-mediated RNA decay (NMD) is an evolutionarily conserved surveillance process that safeguards cells against RNAs that carry nonsense mutations and their truncated translation products. During NMD, a ribosome engaged in translating an aberrant mRNA terminates prematurely. Factors downstream of the prematurely terminating ribosome recruit NMD machinery and initiate destruction of the aberrant mRNA. As a result, nonsense mutations often render a gene nonfunctional due to the rapid degradation of its mRNA transcripts. We and others have shown that the efficiency of NMD varies widely across different genetic and cellular contexts, but the mechanisms that cause this variability remain poorly understood. By investigating nonsense genetic variants that have risen to high frequency in the healthy human population, I have demonstrated that over half of these common nonsense variants escape NMD via mechanisms that include stop codon readthrough and translation reinitiation. These results highlight our incomplete understanding of the range of molecular mechanisms that could rescue gene function in the presence of a predicted ?loss of function? mutation. My research program builds on these novel discoveries to systematically investigate cis and trans factors that allow an mRNA to escape NMD. We will combine massively parallel reporter assays with targeted genetic screens to identify consensus sequence and/or structural features that permit stop codon readthrough and reinitiation. We will complement these approaches with in vivo genome editing to perturb sequence features that promote NMD escape of validated candidates of readthrough or reinitiation. We will use validated sequences that escape NMD to identify trans factors via genetic screens and/or affinity isolation followed by mass spectrometry. Given the role of NMD in shaping gene expression, understanding the mechanisms that cause NMD efficiency to vary has immense practical significance in both precision medicine initiatives and in basic biomedical research.